Overexpression of regucalcin suppresses cell death and apoptosis in cloned rat hepatoma H4-II-E cells induced by lipopolysaccharide, PD 98059, dibucaine, or Bay K 8644
Overexpression of regucalcin suppresses cell death and apoptosis in cloned rat hepatoma H4-II-E cells induced by lipopolysaccharide, PD 98059, dibucaine, or Bay K 8644
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DOI：
10.1002/jcb.20214
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发表时间：
2004-10-15
影响因子：
4
通讯作者：
Yamaguchi, M
中科院分区：
文献类型：
--
作者：
Izumi, T;Yamaguchi, M
本研究利用高表达regucalcin的大鼠肝癌H4-II-E细胞株，研究了regucalcin对细胞死亡的影响。将肝癌细胞（野生型）和稳定regucalcin（RC）/pCXN 2转染子在含有10%胎牛血清（FBS）的培养基中培养72 h以获得亚融合单层。培养72小时后，将细胞在不含FBS且含有载体或脂多糖（LPS; 0.1或1.0 μ g/ml）的培养基中进一步培养12-72小时。在LPS（0.1或1.0 μ g/ml）存在下培养24或48小时，野生型细胞的数量显著减少。LPS（0.1或1.0 μ g/ml）在减少肝癌细胞数量中的作用在过表达regucalcin的转染子中被显著阻止。然而，用LPS（0.1或1.0 μ g/ml）培养72小时导致转染子的细胞数显著减少。用LPS（0.0 μ g/ ml）培养野生型细胞24-72小时，可显著降低Ca 2 +/钙调蛋白依赖的一氧化氮（NO）合酶活性。这种下降在转染子中被显著阻止。LPS（0.1或1.0 μ g/ml）诱导的野生型细胞数量的减少可通过与胱天蛋白酶-3抑制剂（10（-8）M）一起培养而显著防止。此外，通过与PD 98059（10（-6）M）、地布卡因（10（-6）M）或星形孢菌素（10（-6）M）（其是多种蛋白激酶的抑制剂）一起培养，野生型细胞的数量显著减少。在转染子中未观察到PD 98059或地布卡因对野生型细胞数量的影响，但在转染子中观察到星形孢菌素的影响。用Bay K 8644（2.5 × 10（-6）M）（细胞内Ca 2+进入的激动剂）培养，导致野生型细胞数量显著减少。在转染子中未观察到这种效应。在Bay K8644存在下，LPS的存在并没有显著降低野生型细胞的数量。琼脂糖凝胶电泳显示，存在低分子量的脱氧核糖核酸（DNA）片段的粘附野生型细胞与湾K 8644培养，并在转染子中显着防止这种DNA片段。这项研究表明，regucalcin的过表达对LPS或各种细胞内信号相关因子诱导的细胞死亡具有抑制作用。
The effect of regucalcin, a regulatory protein in intracellular signaling pathway, on cell death was investigated by using the cloned rat hepatoma H4-II-E cells overexpressing regucalcin. The hepatoma cells (wild-type) and stable regucalcin (RC)/pCXN2 transfectants were cultured for 72 h in medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured for 12-72 h in medium without FBS containing either vehicle or lipopolysaccharide (LPS; 0.1 or 1.0 mug/ml). The number of wild-type cells was significantly decreased by culture for 24 or 48 h in the presence of LPS (0.1 or 1.0 mug/ml). The effect of LPS (0.1 or 1.0 mug/ml) in decreasing the number of hepatoma cells was significantly prevented in transfectants overexpressing regucalcin. However, the culture with LPS (0.1 or 1.0 mug/ml) for 72 h caused a significant decrease in cell number of transfectants. Ca2+/calmodulin-dependent nitric oxide (NO) synthase activity was significantly decreased by culture with LPS 0.0 mug/ ml) for 24-72 h of wild-type cells. This decrease was significantly prevented in transfectants. LPS (0.1 or 1.0 mug/ml)induced decrease in the number of wild-type cells was significantly prevented by culture with caspase-3 inhibitor (10(-8) M). Moreover, the number of wild-type cells was significantly decreased by culture with PD 98059 (10(-6) M), dibucaine (10(-6) M), or staurosporine (10(-6) M), which is an inhibitor of various protein kinases. The effect of PD 98059 or dibucaine on the number of wild-type cells was not observed in transfectants, although the effect of staurosporine was seen in transfectants. Culture with Bay K 8644 (2.5 x 10(-6) M), an agonist of Ca2+ entry in cells, caused a significant decrease in the number of wild-type cells. Such an effect was not seen in transfectants. The presence of LPS did not significantly decrease the number of wild-type cells in the presence of Bay K8644. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with Bay K 8644, and this DNA fragmentation was significantly prevented in transfectants. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell death induced by LPS or various intracellular signaling-related factors.